14alpha-hydroxy pregnatrienes



UnitedStates Patent ice 14a-HYDROXY PREGNATRIENES David H. Gould,Leonia, Emanuel B. Hershberg, West Orange, and Elliot Shapiro,Irvington, N.J., assignors to Schering Corporation, Bloomfield, N.J., acorporation of New Jersey No Drawing. Application April 30, 1956 SerialNo. 581,265

20 Claims. (Cl. 260397.45)

This invention relates to a new group of steroid compounds and tomethods for their manufacture. More particularly, our invention relatesto oxygenated pregnatetraenes which are potent hormone-like substances.

In this application we employ the accepted terminology for identifyingthe carbon atoms of a pregnane and reproduce below the10513-dimethylpregnane skeleton.

The compounds of our invention are represented by unsaturated pregnaneshaving the following general formula CH OR wherein X is a member of thegroup consisting of (H,aOR), (H,;8OR), and O; R is a member of the groupconsisting of H and acyl; Z- denotes the arrangement of C-8, C-9, and0-14 consisting of and W is a member of the group consisting of H, F, Cland Br.

The compounds'of the general formula which are devoid of a halogen groupat C-9' exhibit an interesting distribution of pharmacologicalproperties. In general, those compounds possessing an OH group at C 17together with either a 50H or keto function at C-ll are adrenocorticoidin their action. The compounds are useful in the-treatment ofinflammation, arthritic discases and certain other collagen diseasessuch as :disseminated lupus, polyarteritis and the like, and areadvantageously employed over known corticoids, such as corti- 2,900,400Patented Au 1-8', 1959.

some and desoxycorticosterone in that they are relatively devoid ofelectrolyte-retention. Thus, following administration of thesepregnatetraenes' there is relatively little sodium retention andconcomitant edema, which is so common with many of the adrenocorticalhormones and corticoids heretofore in use. In combination withmineralocorticoids such as desoxycorticosterone, our compounds provideand enhance resistance to stress and aid in the control of carbohydratemetabolism. Corollary. to the foregoing, these pregna tetraenes areuseful in sup pression of pituitary ACTH output.

A further important application of these new steroids is inthe'treatment of allergic phenomena such as asthma, and certain types ofperennial rhinitis, angioneurotic edema, and drug sensitivities, whichdo not respond to usual antihistaminic therapy. Moderate doses ofthesesubstances are effective in giving prompt relief. to an acute asthmaticattack, or resistant status asthmaticus, which might otherwise provefatal.

The S ot-halogenated compounds of the general formula are useful inmaintaining bilateral adrenalectomi'zed patients and Addisonians.

Topical preparations of the compounds of the invention in the form ofointments, suspensions and the like, lend themselves to ophthalmologicaluse where they are used to block inflammatory reactions arising from alelergic disorders, as well as bacterial or toxic origins (such as acuteiritis).

Those compounds of the general formula possessing an acyloxy group at0-17, especially in combination with a C-11 ester function and/or a C-21ester group such as 1,4,6,8(9) pregnatetraene-1l,B,17a,21-triol 3,20'dionell-formate-(or acetate)-l7-acetate and the corresponding C-21esters, are progestational agents showing 0.5'- 1.5 times the activityof progesterone.

Our new substances may be administered for their particular therapeuticuse either orally or parenterally'in the: form of tablets, capsules,aqueous or oil suspensions or? solutions. In the usual corticoidtreatment such as antitinflammatory therapy, replacement therapy, andthelike, the daily dose is 5-10 mg. depending upon the extent of therapyrequired. In the treatment of allergic phenompionate are particularlyefiective in this instance. Furthermore, certain aryloxy alkanoic acidradicals and certain heterocyclic carboxylic acid radicals, whenattached in the form of an ester link at C-21, provide for a longerduration of activity than is obtained with, the lower alkanoic acidradicals, and thus permit the utili2ation of a smaller does to achieve adesired result. By way of example, such radicals include phenoxyacetate,.substituted phenoxyacetates such as 4-bromo-, 4-methyl-, 4-tertiarybutyl-, 2,4,5-trichloroand 4-methoxy-phenoxyacetates. As examples of theheterocyclic carboxylic acid radicals, we have found that the furoatesand sub-' stituted furoates including the 5-bromo-, 5-met hyl-, andS-tertiary-butylfuroate radicals are especially valuable.

Since the unesterified compounds of the formula together with theirmonobasic acid esters are essentially insoluble in water, we canovercome this difliculty and can provide for water solubilityby formingnon-toxicmetal salts such as the sodium salts of monoesters ofpolycarboxylic acids such as. succinic, phthalic, tricarbalylic,itaconic acids, etc., preferably at 0-21.

r 2,900,400 I l As indicated above, we envision as falling within our Weprefer, however,'to use a halogen'such as chlorine,

invention the I la-OH compounds of the formula bromine, or iodine as theanionic substituent at C-9 OHaOR and we also prefer the chemicalapproach to the overall synthesis. This preferred process is shown inthe following equation x GH OAG w i=0 l -on o EOAo wherein W, X and Rhave the same designation as given. Br T The 14a-hydroxylated steroidsexhibit the aforementioned o therapeutic properties and differ from thepregnatetraenes v Pharmacologically more in degree than in kind. More OHon important, these 14et-hydroxylated compounds are important andnecessary intermediates in the synthesis of =0 the corresponding1,4,6,8(l4)-pregnatetraenes. m 0H The 1,4,6,8(9)-pregnatetraenes of ourinvention are HO Ho preferably prepared from1,4,6-pregnatriene-17u,2l-diol- 3,20-diones which possess an oxygenfunction at C-11. The 1,4,6-pregnatrienes used as starting materials aresynthesized according to the procedure described in co- E6]; pendingapplication of Gould and Herzog, Serial No. 0 513,901, filed June 7,1955. After substitution of an m In anionic radical such as hydroxyl,halide, sulfate, sufonate, nitrate, etc., for an hydrogen atom on either0-8 or 0-9, The stamng compound V m thls mstance mdlcated as thesubsequent elimination of a proton with the anion a 9wbf0m0 bs ance mayalso be a 9a-ch1oro or 9::- gives rise to the pregnatetraenes of thisinvention. Thus, fluor-oanalogi 15 piepared accoralpg to the.amilogousfor example, incubation of 1,4,6-pregnatriene-17,21- Pmcedure descnbed mthe copendmg apphcauon of diol-3,11,20-trione with a microorganism whichis known Gould P 4 i Serial 9 filed June 1955' to hydroxylate at the,C-8 or C-9; position, such as The ,ehmmaum? of hydrohahc acid between 8and H elicostylum pir'iforme or Neurospora crassa, as described 9' 1Sefiected m a known manner deliydmhalogena' in'the copending applicationof Hershberg and Herzog, tron (such as by heating the 9u-halotriene mthe presence Serial'No. 486,028, filed February 3, 1955, gives rise tomm acid.) Elves 1 the corresponding 9) hydroxy derivative The 11B,17et,21-tr1ol-3,20-dione 21-acetate (1b). Sapomficaexact position atwhich hydroxylation occurs is unknown. 9 the t {easily acPoniphshed bytreatment Howeverior the purposes of this process, it is unim 40 withdilute alkali as indicated, yielding Ic. Alternatively,

portant, since the intermediary compound so obtained thedehydmhalogeliaiion may 9 efiected Y Odie: fromemicrobiologicalprocedure is dehydrated so as to agents such as collidine or sodiumacetate 1n acetic acid introduce an additional double bond between 0-8and f Q- d d 0-9. The dehydration is easily accomplished chemically eave a so Dun t at t e compmm S escn e m copending application, SerialNo. 513,901, filed June 7,

by a variety of agents Such as alumna morgamc bases 1955, are usefulintermediates in the preparation of the (sodium hydroxide, sodiumbicarbonate, etc.), aqueous acetic acid, p-toluenesulfonic acid inchloroform or by prlegnatetralelnei-of i g i m thattthey 12 grheating inxylene. This sequence of reactions is shown F? to t 5 t? Ogen a e in thefollowing schematic diagram. (The uncertainty of Posmon as S own m e oowmg equa on the point of attachment of the hydroxyl group introducedCHIOAB by the microorganism is indicated by the use of a bracket 4indicating that attachment may be at either (3-8 or 0-9). --OH 0-(IIHaOH 0H l Bri or NBS 0 f' v v VI ii'fi onioae emote p rllorme 5 o 0-onion V t to: N CA a G t OK HOAO o- Br' r v11 7 Id 1 H0 A] In theforegoing a 21-ester such as the acetate of 1,4,6 0- V :i:fi 0pregnatriene-17ot,21-diol-3,11,20-trioue is reacted with a sul o c c din source of positive halogen ion such as bromine or N-bro- 7 III CHOL,or heatin g xylene medium) mosuccinimide'wherebya bromine atom isintroduced at C-8 (VII). Dehydro'halogenation with sodium acetate inacetic acid, for example, gives rise to 1,4, 6 ,8(9)-pregnatetraene-l7u,21-diol 3, l 1,2 0-trione 2l-acetat'e.

If, in this sequence of reactions, the starting material is the 11 fihydroxy analog of VI, we prefer to first formylate the 11,8-hydroxylgroup with formic acid, according to the procedure described by Oliveto,et al., in the copending application, Serial No. 421,948, filed April 8,1954. After dehydrohalogenation, one obtains the corresponding 1,4,6,8(9)-pregnatetraene containing an 11,8- forrnyloxy group. Saponification iseasily eiifected by means of alkali or microbiologically, usingFlavobacterium dehydrogenans var. hydrolyticumas described in thecopending application of Charney, Serial No. 458,661, filed September27, 1954, now abandoned.

We have found that when the anionic substitutent is adjacent to anactivated hydrogen atom, it is easily reproved with the said hydrogenatom by relatively mild treatment with acid or base which may beaccompanied by heating. For example, reagents such as acetic or formicacids, sodium acetate, alkali bicarbonates, carbonates or heating ininert solvents are useful in effecting this dehydrohalogenation. On theother hand, when the substituent is not adjacent to an activatedhydrogen atom such as would be in the situation when the startingmaterial is 1,4,6-pregnat1iene-1 1,9,17a,2l-triol-3,20-dione-llformate-Zl-acetate, somewhat stronger measures toeffectdehydrohalogenation are necessary. Methods for effect- .ing thistype of dehydrohalogenation are exemplified by dehydration with a strongacid; moderate pyrolytic procedures in the presence of organic basessuch as collidine, quinoline, etc., at 150-250"; treatment with strongdehydrohalogenators such as silver or mercury salts in organic solventssuch as pyridine, collidine, acetonitrile, etc.

3 In order to prepare the 1,4,6,8(14)-pregnatetraenes of the generalformula, we prefer tohydroxylate a 1,4,6- pregnatriene at the 0-14position, isolate the 14u-hydroxy compound and dehydrate to thetetraene. The following sequence of reactions illustrates a method forpreparting the 14m-hydroxy intermediates .CHaOH e ----0H .W

/ Curvularla '0 lunata VIII $HOA0 C110 --,OH CHaCO Cl I pyridine w Inthe above reaction apregnatriene (VIII) is subjected to the oxygenatingactivity of a microorganism which hydroxylates in the Mot-position suchas Curv ulari'a lunata. The productso formed is the corresponding 14uhydroxyp-regnatriene, IX, which may be esterified at C-21 with acetylchloride in pyridine, for example;

Alternatively a 4,6-pregnadiene such as 4,6-pregna- -diene-17a,21-diol-3,l1,20-trione may be hydroxylated with C zrvularialunaja so asto producethe corresponding 14d-hydroxy-pr'egnadiene; subjecting thelatter comr .6 pound to the dehydrogenating action of a dehydrogenatingorganism, -such-as C. simplex or B. vsphaerip us affords 1,4,6pregnatrienee14 t, 17a,21-triol-3,11,20-tri0ne. Conversion of the14a-hydroxy compounds to 1,4,6, 8(14)-pregnatetraenes is carried out ina facile manner. In general, the major step is the removal of an anionicsubstituent at C14 together With the hydrogen atom at C8 which isactivated by the conjugated unsaturation in rings A and B, leading tothe insertion of a double bond between 0-8 and C-14 which is inconjugation with the previous unsaturation. We generally prefer as theanionic substituent a 14u-hydroxy1 group.

Acetyl chloride w Pyridine As dehydration procedures there are used thefollowing: saponification or treatment with mild base; treat.- ment withacid, particularly warming with dilute organic or weak acids;chromatography or other treatment with alumina, thoria and the like;esterification with acidic agents, e.g. acetic anhydride; andthermal'splitting, e.g. by heating in toluene, xylene, collidine and thelike. It is to be noted that stronger treatment with acid or .base inthe case of 1,4,6,8(14)-pregnatetraene-17a,21ediol-3,11, 20.-trioneisomerizes the terminal double bond to give 1,4,6,8 (9-pregnatetraene-17a,21 diol-3, 1 1,20-trione. I

Partially esterified p-regnatetraenes may be prepared ac? cording to avariety of prescribed procedures. .We prefer .in general to fullesterify all hydroxyl groups which are present and then selectivelysaponi fy as required. For example, 1,4,6,8 (9) -pregnatetraene-1 113,:,2 1-triol -3,2.(:).- dione may be acylated with reagents such asacetic anhydride and acetic acid preferably in the presence of p-toluenesulfonic acid whereby acetylation occurs at the 11,17 and '21-positionaffording the corresponding triacetate. Subjecting the triacetate .soobtained to mild hydrolytic conditions results in the formation of thecorresponding 11fl,17o,21-tri0l-1;l,17-diacetate. Instead of theaforementioned acylating agent, other agents such as formic acid in thepresence of p-toluenesulfonic acid are applicable and the correspondingformate esters are obtained.

An ll-keto 17,21 diester may be reduced either chemically ormicrobiologically according to known methods whereby the corresponding11p-hydroxy- 17,21di ester is produced. Other methodsof preparingvarious combina: tions are set forth in the following examples.

The following examples arepresented to'illustrate various methods forpreparing the compounds of ourv inyene tion. It will be apparent to achemist skilled in the art that various modificationsrare inessence nomore equivalents. Thus our invention is limited only as finedintheappen'ding'claims;

..EXAMPLE.1-

1,51,6,8-1zregndtetraene-17a,21-di0l-3,11,20-tri0ne ZI-acez'ate j e (A)A mixture of 500 mg. of9a-chloro-1,4,6-pregnatriene-17a,21-diol-3,-l1,20-trione 21-acetate(prepared by substituting hydrogenchloride for hydrogen fluoride in theprocedure described in the copending application of Gould and Herzog,Serial No. 513,901, filed June 7, 1955) and 10 m1. of 'y-collidine isrefluxed for 15 minutes. The cooled mixture is poured into excesssulfuric acid in ice, and extracted with methylene chloride. The organiclayer is washed with water until neutral, dried and concentrated to asmall volume. The concentrate is chromatographed on activated magnesiumsilicate and fixed by using hexane as the developer. Chromatographicelution with ether-hexane mixtures aifords the compound of this examplein the fraction where ether is the eluting solvent. Purification iseffected by recrystallization from acetone hexane.

(B) Alternatively, the compound of this example may be prepared bytreating a solution of 300 mg. of 9a.- bromo 1,4,6 pregnatriene 176:,21dial-3,11,20-trione- 3,20-acetate in 5 ml. of chloroform and 10 ml. ofacetic acid with 56 mg. of anhydrous sodium acetate in 5 ml.

of acetic acid. The mixture is refluxed for two hours, concentrated invacuo and poured into water. The resultant precipitate from methylenechloride-hexane affords the tetraene of this example.

(C) A further procedure for the preparation for the compound of thisexample is as follows: To a stirred solution of 1.0 g. of1,4,6-pregnatriene 170:,21- diol- 3,11,20-trione-21-acetate in '10 ml.of methylene chloride is dropwise added 0.22 g. of bromine in 5 ml. ofacetic acid at a rate governed by the disappearance of color due toexcess bromine. After an additional 15 minutes stirring, thesolution isconcentrated in vacuo to remove methylene chloride and the residualmixture is poured into water. --The solid is removed by filtration,washed with water, dried, and crystallized from acetone-hexane to givethe intermediate 8-(or 9-)bromo-1,4,6-pregnatriene-17e,21-diol-3,11,20-trione ZI-acetate.

The intermediary 8-bromo-1,4,6-pregnatriene-17,21-diol-3;11,20-trione.'21-acetate may. alternatively be 'preparedaccording to the following:

'A sample. of .1 g. of 1,4,6-pregnatriene-17a,2Ldiol- 3,11,20-trione 21-acetate is dissolved in 50 of a 1:1 mixture of carbon tetrachloride andchlorobenzene. The solution is brought to a boil by irradiation with a300 w.

photoflood lamp and treated with 0.40 g. of N-bromo:

succinimide. The mixture is boiled with irradiation for 10-15- minutesuntil the solution no longer gives a starch iodide test. T he mixture iscooled, filtered, washed with water and 'e vaporatedjto drynessin vacuo.Crystallization from methylenechloride-hexane afiords the 8- (or 9-)bromo-1,4,6-pregnatriene-17 ,21-diol-3,11,20 trione 21 -acetate. The8-bromopregnatriene intermediate so obtained is dehydrohalogenated withsodium acetate in acetic acid according to the procedure described inpart B yielding 1,4,6,8-pregnatetraene 17:,21 diol-3,11,20- trione21-acetate.'

, r EXAMPLE 2.-

g 1,4,6,8-pregnatetraene-1 7oz,Z1-di0l-3,1 1,20-tri0ne Nitrogen gas ispassed through a solution of 50 mg. of the e'ster obtained in Example 1'i115 ml. of methanol so as to remove .dissolved air. .To the methanolicacid is added mg. of potassium bicarbonate in 0.5 ml. of waterand theresulting mixture is allowed to stand in an atmosphere of nitrogenovernight. Dilution of'the mix tore-with water affords a crystallinematerial which is removed by'filtration andfdried. Recrystallizationfrom aqueous-acetoneafiords thecompound of this example.

EXAMPLE 3 1,4,6,8-pregncitetraene-1148,17a,2I-tri0l-3,20-i1i0ne 21-acetate (A) A sample of 0.2 g. of9a-bromo-1,4,6-pregnatriene-l1p,l7a,21-triol-3,20-dione 21-acetate isdissolved in 10 ml. of acetic acid containing 0.4 g. of sodium acetateand the solution is boiled 30 minutes. The solution is cooled and pouredinto water and the separated solid is extracted with methylene chloride.The organic layer is washed with aqueous sodium bicarbonate and watertill neutral, and dried with anhydrous magnesium sulfate. The driedsolution is filtered, concentrated and chromatographed on activatedmagnesium silicate. The fraction eluted with 10-25% methylene chloridein ether is crystallized from ethyl acetate to givel,4,6,8-pregnatetraenellfi, l7a,2l-triol 3,20-dione ZI-acetate.

(B) Alternatively, the compound of this example is prepared in thefollowing manner. One gram of 1,4,6-pregnatriene-l1,8,17a,2l-trio1-3,20-dione ll-formate 21- acetate isbrominated with 0.21 g. of bromine and the product is crystallized fromaqueous acetone to give 8-bromo-1,4,6-pregnatriene-11 8,17a,2l-triol3,20 dione ll-formate 21-acetate according to the procedure described inExample l-C.

A sample of 0.8 g. of the bromopregnatriene so obtained is, dissolved in20 ml. of 'y-collidine and treated with 1.5 g. of silver nitrate. Thesolution is warmed on the steam bath at -95 for 6 hours and poured intoice water. The mixture is acidified with dilute sulfuric acid andextracted with methylene chloride. The combined methylene chloridewashes are washed neutral with water, dried over magnesium sulfate,filtered and evaporated. The residue is crystallized several times fromacetone-hexaneto give 1,4,6,8 pregnatetraene 11,8,17iz,2ltr-iol-3,20-dione 11- formate Zl-acetate.

EXAMPLE 4 V 1,4,6,8-pregnatetraene-11 3,17%21 -triol3,20-dione A sampleof 0.05 g. of the product of Example 3 is saponified and crystallized asin Example 2 to givel,4,6,8-pregnatetraene-l118,l7a,21-triol-3,20-dione.

Alternatively, the following process is applicable;

A mixture is prepared of 1 g. ofyeast extract concentrate and 1 ml. eachof 2 M potassium dihydrogen phosphate and 2 M disodium phosphate in eachml. Ten Erlenmeyer flasks (300 ml.) containing 100 ml. each aresterilized and innoculated with Flavobacterium dehydrogenans varhydrolyticum according to the procedure described in copendingapplication of William Charney, Serial No. 458,661, filed September 27,1954. The flasks are shaken at 30 for 16 hours, and to each is added asolution of 50 mg. of 1,4,6-pregnatriene-11,17a,21-triol- 3,20-dionell-formate 21-acetate in 5 ml. of methanol. The cultures are shaken at30 for 24 hours and the combined broths are extracted three times with300 ml. of methylene chloride and the extract is dried, filtered andevaporated to dryness. The residue is crystallized from acetone to givel,4,6,8-pregnatetraene-l lfl,17a,21-triol- 3,20-dione.

EXAMPLE 5 1,4,6,8 (9) -pregnatetraene-1 15,1 70:,21 -triol-3,20-

' a dione triacetate night and'upon dilution with water, the triacetateprecipitates. The ester is separated by filtration and recrystallizedfrom ether-hexane solvent, aifording 1,4,35,8(9); B -s am e elislamis iii iilii t r 9 EXAMPLE 6 1,4,6,8 (9) -pregnatetraene-I 7a,.21 -dil-3,11,ZO-dione diacetate A mixture of 500 mg. of the compound of Example 2 in5 ml. of acetic ahhydride and 5 ml. of acetic acid and 50 mg. ofp-toluenesulfonic acid is processed according to the procedure describedin Example 5. Recrystallization from ether-hexane mixtures affords thediacetate of this example.

EXAMPLE 7 1,4,6,8 (9) -pregnatetraene-11fl,1 70:,21 -tri0l-3,20- dionetriformate To a mixture of 10 ml. of formic acid and 1 1g. of the tn'olof Example 4 is added 100 mg. of p-toluenesulfonic acid. The mixture ischilled in a refrigerator for 48 hours after which time it is dilutedwith water and the resultant precipitate removed by filtration. Thecrude triformate so obtained is purified by recrystallization fromether-hexane.

EXAMPLE 8 I 1,4,6,8 (9) -pregnatet raene-11fi,17ot,21 -triol-3,20-

dione 11,17-diformate EXAMPLE 9J,4,6-pregnatriene-14a,1701,21-di0l-3,11,20-trion and1,4,6-pregnatriene-8d1711,21-triol-3,1l,20-tri0ne One hundred grams of.edamine enzymatic digest of lactalbumin, 15 g. of corn steep liquor and250 g. of cerelose is diluted to 5- l. with ,tap water and adjusted withsoda to pH 4.3-4.5. The medium is divided into twenty-five 500 m1.flasks and sterilized by heat, cooled and inoculated with a culture ofHelicostylum pirifprmc, A.T.C.C. No. 8992. The flasks are incubated 24hours at 28 while shaking on a shake table. To each fiask is added asolution of 50 mg. of 1,4,6-pregnatriene-17,2ldiol-3,11,20-tn'one(prepared according to the procedure described in copending applicationof Gould and Herzog, Serial No. 513,901, filed June 7,1955) dissolved in.a minimum amount of acetone. The flasks are again shaken and incubatedat 28 for 24 hours, after which 50 ml. of methylene chloride is added toeach flask. After shaking, the organic layer is separated and combinedwith a second methylene chloride wash. The combined solutions are dried,filtered and evaporated to .a. crystalline residue. This residue istriturated with hexane and the hexane withdrawn. The remainder isdissolved in a minimum amount of methylene chloride (30-50 ml.), andchromatographed on 50 g. ofactivated magnesium silicate. The column iswashed with 500 ml. of anhydrous ether, and 1 l. of methylene chloridefollowed byv five 200 ml. fractions of methylene chloride containing0.5% methanol and five fractions containing 1% methanol. Elution of thedesired product is obtained with 1.53% methanol in methylene chloride.

Crystallization of the residue obtained on evaporation fromacetone-hexanegives 1,4,6-pregnatriene-14a,1712,21-triol-3,l1,20-trione. "Further elution with 3-5% methalnol in methylenechloride gives a diflerent product, 1,4,6-

'pregnatriene-BB,17a,21-trio1-3,l1,20-trione, also crystal- 'lizablefrom acetonehexane. Y

' '10. EXAMPLE 10 1,4,6.-pregnatri e'ne-14a,] 7a,21-triolF3,11,20vtrione21-propionate A sample of 0.1 g. of the 140c-l'1YdI'0XY product ofExample 9 is dissolved in 5 ml. of dry pyridine and chilled to 5. Thissolution is treated with 50 mg. of propionyl chloride with stirring at 0for 15 minutes and at 15 for 15 minutes. The mixture is poured into 50ml. of water and extracted with methylene chloride. The organic layer iswashed with dilute hydrochloric acid, dilute sodium bicarbonate andwater until neutral. The dried solution is filtered and evaporated to aresidue which is crystallized from aqueous acetone togive1,4,6-pregnatriene-14a,17a,21-triol-3,11,20-trione 2l-propionate.

EXAMPLE 11 1,4,6-pregmzzriene 11;8,140z,1 7 0c,21 -tetr0l-3,20 dione and1,4,6-pregnatriene-8/9,115,1 7u,21-tetrol-3,20-di0ne- The procedure ofExample 9 is .repeated using as substrate1,4,6-pregnatriene-11B,17a,21-triol-3,20-dione in the same amounts. Theresidue is chromatographed as above and washing with 24% methanol inmethylene chloride elutes fractions which on evaporation andcrystallization from methylene chloride-hexane give the desired product,1,4,6-pregnatriene-l1B,l4a,17a,21 tetrol-3, ZO-dione. Further washingwith 4-6% methanol in methylene chloride elutes crystalline materialwhich on crystallization from methylene chloride-hexane gives 1,4,6-tpregnatriene-8,8, 1 1B,17a,21-tetrol-3 ,20-dione.

EXAMPLE l2 1,4,6-pregnatriene-11fi,14 x,17a,21-tetrol-3,20-di0ne21-capr0ate I Using the procedure of Example 10 with the 14ot-hydroxycompound of Example 11 and caproyl chloride, the desired product isobtained. It is 'crystallizable hem methanol and aqueous acetone.

EXAMPLE 13 9a-flu0r0-1,4,6-pregnatriene-14a,1701,21-trial-3,11-20-tri0ne at C. and the cooled broth in each flask is inocu-.lated with 1 ml. of a suspension of Curvularia lunqta from a 24-hourbroth culture. The mixtures are incubated on a shake table for 20 hoursat 28 C. After incubation each flask is treated with a solutioncontaining 50 mg. of sterile 9a-fluoro-1,4,6-pregnatriene-170:,21-.diol-3,ll,20-trione in 5 ml. of ethanol. The pH is 6.8-7.2. Thecultures are then incubated and shaken for 48 hours at 28 C. The pH is7.0-7.4. The cultures are then extracted thoroughly with chloroform. Theextracts are combined and evaporated to dryness, giving a residue of ca.500-600 mg.

The crudeuextract is triturated with methanol giving a crystalline solidwhich is crystallized from acetoneto give 9a fluoro-l,4,6 pregnatriene14d,170t,21 triol- 3,11,20-trione.

EXAMPLE 14 Example 13 is repeated using as substrate 9a-fluoro-1,4,6-pregnatriene-11,8,170 2l triol-3,20 dione. The productiscrystallized from methylene chloride-hexane to give '9a-fluoro 1,4,6pregnatriene 11,18,14a, 1'7a,2'ltetrol-3,20-dione.

. 1,4,6,8(14) pregnatetraene 170:,21 diol-3,11,20-trione e 11 E AMP EUsing the procedure of Example 13 with 9m-bromo- 1,4,6-pregnatriene1113,17a,21 triol 3,20 dione, a residue is obtainedwhich is crystallizedfrom acetone hexane to give'9u bromo 1,4,6 pregnatriene 11B,

. 14m,17a,21-tetro1-3,20-dione.

' EXAMPLE 16' EXAMPLE l7 ZI-abetates of l4u-hydr0xy compounds 'Using theprocedure of Example .10 with acetic anhydude in place of propionylchloride, a residue is obtained, which gives on crystallization frommethanol ;1',4,6-- pregnatriene 14a,l7cc,21 triol 3,11,20 trioneill-acetate. V

The same procedure applied to the 14a-hydroxy compound of Examplellgives on crystallization from acetone 1,4,6 pregnatrien 11B,14a,17a,21tetrol 3,20- dione ZI-acetate.

The same procedure with the product of Example 13 gives oncrystallization from acetone-heXane9a-fluoro- 1,4,6 pregnatriene14a,17dz,21 triol 3,11,20 trione ZI-acetate. i The same procedure withthe product of Example 14 gives on crystallization from acetone9a-'fluoro-l,4,6- pregnatriene 1113,14c,17oz,21 tetrol 3,20-dione-21-acetate.

EXAMPLE 1s 1,4,6,8( 14)-firegnaletraene-I15J7 ,21-triol3,20-dine 1,4,6pregnatriene 11fi,14a,l7oc,21 tetrol 3,20- dione (0.5 g.) is dissolvedin 20 ml. of methanol and nitrogen is bubbled through the solution. Tothis is added'a solution of 0:14 g. of potassium bicarbonate in 1 ml. ofwater and the mixture is stirred for eight hours at room temperature.The solution is concentrated in vacuo and addition of water givescrystals which are collected and dried. Recrystallization fromacetone-hexane gives 1,4,6,8(14) pregnatetraene 11B,l7a,21 triol-3,20-dione.

The same product is obtained by the same procedure when 0.5 g. of1,4,6-pregnatriene-11p,14a,17a,21 tetrol 3,20-dione 2'l-acetate is usedas the starting material.

EXAMPLE 19 1,4,6,8 (14 pregnatetraene-1 7a,21-di0l-3,11 ,2 O-trionepregnatriene 14Dt,170t,2.i' triol 3,11,20 trione 21- acetate, dilutionwith Water gives a product which is collected, dried, recrystallizedfrom methanol to-give ZI acet-ate. V V

.2 EXAMPLE so triol-3,20ldiorte A same of 0,5 g. of9a-fluoro-1,4,6-pregnatriene-11 9,

14oz,17a,21-tetro1-3,20-diqne 21-acetate is treated as in Example 19leading to a product which, on recrystallization from methanol gives9a-fluoro-1,4,6,8(14)- pregnatet-raene 11,6,17a,21 triol 3,20 dione21-acetate. i

A sample of 0.2 g. of this substance isdissolved in 8 m1. of methanoland nitrogen is'bubbled through. To the solution is added 0.53 g. ofpotassium bicarbonate in 0.53 ml. of water and the mixture is maintainedunder nitrogen for 4- hou'rs. Dilution with water gives a solidawhich iscollected, dried and recrystallized from aqueous acetone to give9a-fluoro-1,4,6,8(14)-pregnatetraen'e-l15,17a,21-triol-3,20-dione.

EXAMPLE 21 9u-br0m0-1,4,6,8(14)-pregnatetraene-l1B,17a,2i-

triol-3,20-diane 21 -acetate A sample of 0.4 g. of9a-bromo-1,4,6-pregnatI-iene- 11 8,14 ,17a,21-tetrol-3,20-dione isdissolved in 4 ml. of acetic anhydride while heating to -100" on a steambath for 1 hour. The'solution is cooled, treated with 4 ml. of methanoland poured into' 40 m1. of water. The precipitate is filtered ofii,dried and recrystallized from aqueous methanol to give9a-bromo-1,4,6,8(14)-pregnatetraene-l1B,17a,21-triol-3,20-dione21-acetate.

EXAMPLE 22 9eg-flu0r0-1,4,6,8(14)-pregnatetraene-1 70;,21-dial-3,11,20-tri0ne A sample of 0.2 g. of 9afluoro1,4,6-pregnatriene-14oz,17u,21tri0l-3,11,20-trione is dissolved in 20 ml. of methylenechloride and passed through a 10 cm. column of 20 got acid-washed andactivated alumina (-200 mesh). 1The fraction eluted with 25% methanol inmethylene chloride is evaporated and crystallized from acetone-hexane.to give 9a-flu0ro-1,4,6,8(14)-pregnatet-.raene-17a,21-diol-3,11,20-trioue. i i

When the starting material 's the corresponding '21- acetate, theproduct, which is crystallized from aqueous acetone is'9u-flu0ro1,4,6,8(14)-pregnatetraene-17u,2ldiol-3,11,20-trione21-acetate. Y 1 t EXAMPLE z4 i 1,4,45,8(14)-pregnatetraene-11,3,17m,21ariol-azo-aion 21-(5@terL-butylfiuroate, 21 acetate, and 21 heptanoat Asample .of 0.5 g. of the product of Example, 18 is dissolved in 5 ml. ofdry pyridine and chilled to -5. To this is added 0.3 g. of5-tert.-butylfuroyl chloride and the mixture is stirred 15' minutes at 0and ,30fmijnutes at room temperature. The mixture 'is poured'fintoi ofwater and thedried precipitate is crystallized from'niethanol to givethe desired esterI; 1 l 1 H The same procedure using 0.25 g.OfQ'aceticanhydride in place of tert.-.but'ylfuroyl chloride gives thecorrespond in'g"21-acetate ,whichis recrystallized from methanol. I} Thesame procedure using-0.3 glpf heptanoyl chloride place 'of acetic fanhydride gives the corresponding 21'- heptanoat which isreerystainmfrom aqueous acetone.

13 This application is a continuation-in-part of our application, SerialNo. 559,514, filed on January 17, 1956, now abandoned.

We claim: 1. Unsaturated pregnanes of the formula:

wherein X is chosen from the group consisting of O; and

and R is chosen from the group consisting of hydrogen and a hydrocarbonacyl group having between one and three carbon atoms.

3. 2l-lower alkanoic acid esters of9a-W-1,4,6-pregnatriene-l1;9,14a,17a,2l-tetrol-3,20-dione wherein W ishalogen having an atomic number less than 53.

4. 21-lower alkanoic acid esters of1,4,6-pregnatrienel4cc,l7ot,2lifiOl-3 ,11,20-trione.

5. 1,4,6-pregnatriene-14a,17a,21-trio1-3,11,20-tri0ne.

6. 9a-fluoro-1,4,6-pregnatriene-11fi,14a,17u,21-tetro1-3, 20-dione.

7. 2l-lower alkanoic acid esters of9u-W-1,4,6-pregnatriene-14a,17a,21triol-3,l1,20-trione wherein W ishalogen having an atomic number less than 53.

8. 21-lower alkanoic acid esters of 1,4,6-pregnatriene-11B,140z,170c,21-tetIO1-3 ,20-dione.

9. 9a-W-1,4,6-pregnatriene-1 1,8,140c,17oz,21-iet1'01 3,20- dionewherein W is halogen having an atomic number less than 53.

10. 9e-W-1,4,6-pregnatriene-14a,17u,21-trio1 3,11,20- trione wherein Wis halogen having an atomic number less than 53.

11. 1,4,6-p18g113.tfi6116-140c,170c,21-t1'i01 3,11,20 trione21-propionate.

12. 1,4,6-pregnatriene-14a,17a,21-trio1- 3,11,20 tn'one 21-acetate.

13. 1,4,6-pregnatriene-1 1(3,14a,17oc,21-'et1O1-3,20-diOn6 21-acetate.

14. 9a-fiuoro-1,4,6-pregnatriene-14a,17a,21-trio1 3,11, 20-trione21-acetate.

15. 9a-fluoro-l,4,6-pregnatriene-1 1 8, c,170c,21-tet1'01- 3,20-dione21-acetate.

16. 1,4,6 pregnatriene-116,140,17a,21-tetro1-3,20-dione.

17. 1,4,6-pregnatriene-11,8,14a,17a,21 tetro1-3,20-diOne 21-caproate.

18. 9a-fluoro-1,4,6-pregnatriene-14a,17a,21-triol 3,11, 20-trione.

19. 9a-bromo-1,4,6-pregnatriene-11p,14a,17a,21-tefl'o1- 3,20-dione.

20. 9m-chloro-1,4,6-pregnatriene-14a,17u,21-triol 3,11, ZO-trione.

References Cited in the file of this patent UNITED STATES PATENTSDjerassi Mar. 29, 1955 OTHER REFERENCES

1. UNSATURATED PREGNANES OF THE FORMULA: